H5N1 subtype influenza A virus has evolved into many HA clades since late 1990s. Six circulating H5N1 influenza viruses clustered to a novel branch in clade 2.3.4 and could escape vaccine protection, indicating their antigenic drift. Eleven amino acids substitutions in three antigenic sites of the hemagglutinin of these isolates were found when compared with the hemagglutinin of the primary viruses in clade 2.3.4. On the backbone of the novel isolates A/chicken/Northern China/k0602/2010, we generated a panel of recombinant viruses with HA mutations of restoring the primary vaccine strain Re-5's amino acid and homologous antisera to determine the role of these substitutions. The results of cross-HI assay, micro-neutralization assay and the antigen map of the mutated recombinant viruses showed that three substitutions in antigenic site B, especially D205K, are the major contributors to the antigenic drift of the novel branch of clade 2.3.4. Our study highlights the importance of surveillance of antigenic drift of H5N1 viruses for the control and preparedness of pandemic threats.

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.

Green fluorescent protein (GFP) used as a powerful marker of gene expression in vivo has so far been applied widely in studying the localizations and functions of protein in living cells. In this study, GFP-labeled assay was used to investigate the subcellular localization of matrix (M) protein of different virulence and genotype Newcastle disease virus (NDV) strains. The M protein of ten NDV strains fused with GFP (GFP-M) all showed nuclear-and-nucleolar localization throughout transfection, whereas that of the other two strains were observed in the nucleus and nucleolus early in transfection but in the cytoplasm late in transfection. In addition, mutations to the previously defined nuclear localization signal in the GFP-M fusion protein were studied as well. Single changes at positions 262 and 263 did not affect nuclear localization of M, while changing both of these arginine residues to asparagine caused re-localization of M mainly to the cytoplasm. The GFP-M was validated as a suitable system for studying the subcellular localization of M protein and could be used to assist us in further identifying the signal sequences responsible for the nucleolar localization and cytoplasmic localization of M protein.

ABSTRACT In this study, the complete genomic sequence of a novel reassortant H9N2 avian influenza virus (AIV) from domestic ducks in eastern China was reported. Phylogenetic analysis showed that seven of the eight genes were all highly homologous to the chicken-origin H9N2 viruses, whereas the PB2 gene was homologous to the human-origin H1N1 virus, which suggested that domestic ducks might play a key role in the genetic reassortment and evolution of H9N2 AIVs in eastern China.

One H5N8 and three H5N5 highly pathogenic avian influenza (HPAI) viruses which derived their HA genes from the Asian H5N1 lineage were isolated from poultry during 2009–2010 in mainland China. Pathogenicity studies showed that these viruses were all highly virulent to chickens, while they varied from moderate to high virulence in mice and from mild to intermediate virulence in mallards. Phylogenetic analyses showed that these viruses were reassortants bearing the H5N1 backbone while acquiring PB1, NP and NA genes from unidentified non-H5N1 viruses, and had developed into three distinct genotypes (B–D). Molecular characterization indicated that all these viruses might resist to antiviral agents. Our findings highlight the emergence and development of HPAI H5 viruses of other NA subtypes in H5N1 endemic areas and their potential threat to poultry industry and public health.

ABSTRACT Here, we report the complete genomic sequence of a novel reassortant H4N2 influenza virus isolated from domestic ducks in the Jiangsu province of China in 2011. Phylogenetic analysis showed that all the viral genes except for hemagglutinin (HA) were highly homologous to the clade 2.3.4 H5N2 viruses. The data suggest that genetic reassortment occurred between H4 and H5N2 avian influenza viruses, which highlights the role of domestic poultry as a reassortment vessel in China.

We characterized 7 highly pathogenic avian influenza A(H5N1) viruses isolated from poultry in China during 2009-2012 and found that they belong to clade 2.3.4 but do not fit within the 3 defined subclades. Antigenic drift in subtype H5N1 variants may reduce the efficacy of vaccines designed to control these viruses in poultry.

There is multiple evidence that swine may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of one strain of H6N6 influenza virus isolated from swine with clinical signs of infection in China. Although phylogenetic analysis revealed that this virus might originate from domestic ducks, pathogenicity experiments indicated that the virus replicated in mice without prior adaptation. These findings suggest that the virus has transmitted from ducks to swine in China, and replicated in mammalian hosts without prior adaptation, which may pose a threat to both veterinary and public health.

The role of live bird markets (LBMs) in the perpetuation of avian influenza virus (AIV) has been well studied worldwide; however, there is a paucity of information on the prevalence of different AIV subtypes in LBMs in Eastern China. In this study, long-term surveillance was conducted to investigate the prevalence of AIV in chickens, ducks, and geese in an LBM in Yangzhou city, Jiangsu province, Eastern China, between July 2002 and May 2011. The study used primary virus isolation and subtype-specific reverse-transcription-PCR. In total, 23 different HA-NA subtype combinations were detected, mainly in domestic ducks. This result suggests that domestic ducks play a more important role in LBMs in Eastern China.

According to the cleavge site sequence of Newcastle disease virus（NDV） F gene, a pair of primers and a T aqMan probe were designed and synthesized. A serial 10 fold dilutions positive plasmid was prepared and used for standard. The standard curve revealed the linear relationship between CT （cycle threshold） and template concentration with a good correlation （R2=0.999）. The RRT-PCR method was highly specific and sensitive and could be used for rapid quantitative detection of mesogenic and virulent NDV. No cross-reaction was detected against other avian disease viruses. Sensitivity was 3 copies ofNDV genome. The total meet rate with traditional virus isolation method was about 90.0% in detecting 187 clinical samples as well as 100% in 28 standard isolated strains. It showed that a real-time RT- PCR method for quick, specific sensitive and quantitativedetection of mesogenic and virulent NDV had been established in this study and it displayed good prospects in the rapid screening of clinical samples and epidemiological monitoring of NDV.

In spring 2009, one strain of H5N1 clade 2.3.2 virus was isolated from wild swans in Shanghai, indicating the importance of the wild swan in the ecology of this highly pathogenic avian influenza virus (HPAIV) in Eastern China. Pathogenicity experiments conducted in this study indicated that the virus was highly pathogenic for chickens but lowly pathogenic for mammalian hosts, as evidenced by reduced infection of mice. The analysis of complete genome sequences and genetic evolution showed that A/Swan/Shanghai/10/09 (SW/SH/09) may be derived from the strain A/silky chicken/Shantou/475/2004 (CK/ST/04), which is homologous to the influenza viruses isolated from chicken, duck, pika, little egret, swan, mandarin duck and bar-headed goose in China Hunan, China Qinghai, Mongolia, Russia, Japan, Korea, Laos and Hong Kong during 2007–2011, indicating that the virus has retro-infected diverse wild birds from chicken, and significant spread of the virus is still ongoing through overlapping migratory flyways. On the basis of the molecular analysis, we also found that there was a deletion of the glycosylation site (NSS) in amino acid 156 of the hemagglutinin (HA) protein when compared with that of the other Clade 2.3.2 viruses isolated between 2007 and 2011. More importantly, the sequence analysis of SW/SH/09 virus displayed the drug-resistant mutations on the matrix protein (M2) and neuraminidase (NA) genes.

N-linked glycans are composed of three major types: high-mannose (Man), hybrid or complex. The functional role of hybrid- and complex-type N-glycans in Newcastle disease virus (NDV) infection and fusion was examined in N-acetylglucosaminyltransferase I (GnT I)-deficient Lec1 cells, a mutant Chinese hamster ovary (CHO) cell incapable of synthesizing hybrid- and complex-type N-glycans. We used recombinant NDV expressing green fluorescence protein or red fluorescence protein to monitor NDV infection, syncytium formation and viral yield. Flow cytometry showed that CHO-K1 and Lec1 cells had essentially the same degree of NDV infection. In contrast, Lec2 cells were found to be resistant to NDV infection. Compared with CHO-K1 cells, Lec1 cells were shown to more sensitive to fusion induced by NDV. Viral attachment was found to be comparable in both lines. We found that there were no significant differences in the yield of progeny virus produced by both CHO-K1 and Lec1 cells. Quantitative analysis revealed that NDV infection and fusion in Lec1 cells were also inhibited by treatment with sialidase. Pretreatment of Lec1 cells with Galanthus nivalis agglutinin specific for terminal α1-3-linked Man prior to inoculation with NDV rendered Lec1 cells less sensitive to cell-to-cell fusion compared with mock-treated Lec1 cells. Treatment of CHO-K1 and Lec1 cells with tunicamycin, an inhibitor of N-glycosylation, significantly blocked fusion and infection. In conclusion, our results suggest that hybrid- and complex-type N-glycans are not required for NDV infection and fusion. We propose that high-Man-type N-glycans could play an important role in the cell-to-cell fusion induced by NDV.

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.

To investigate the epidemiology of low pathogenic avian influenza viruses（LPAIVs） in Eastern China,we surveyed LPAIVs in a live-poultry market（LPM） in Eastern China from October 2009 to September 2010,and 58 from 1 650 cloaca swab samples were positive for LPAIVs in isolation when characterized by hemagglutination（HA） and hemagglutination inhibition（HI） tests.These LPAIVs belonged to six HA subtypes,that is H6,H3,H1,H4,H9 and H11,in the order of isolation rate.In addition,seven NA subtypes including N1,N2,N3,N4,N5,N6 and N8 were identified in these LPAIVs.Altogether 11 HA-NA subtype combinations were identified in our study.The isolation rate from domestic ducks was 7.28%,which was much higher than that from chickens（1.00%） and domestic geese（1.02%）.The isolation rate of LPAIVs was distributed with significant seasonal variations,higher during March-June and October-December and lower in the hottest month in summer and coldest month in winter.Six samples were identified to contain viruses of two different HA subtypes simultaneously,accounting for 10.34% of all the positive samples.All six samples of mixed infection were waterfowl-origin.The result indicates that LPM may serve as a gene pool of avian influenza viruses,and domestic waterfowls may act as a reservior of avian influenza viruses,therefore monitoring of avian influenza viruses in LPM,especially in domestic waterfowls,should be reinforced.

For the first time we report the complete genomic sequence of an H11N3 influenza virus from domestic ducks in China. Phylogenetic analysis showed that the H11N3 virus was a novel reassortant with its genes from different subtypes of domestic duck-origin avian influenza viruses, which further underlined that domestic ducks play a key role in the genetic reassortment and evolution of influenza viruses in China.

To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.

To explore the evolutionary patterns of avian influenza virus(AIV) in poultry in Eastern China,epidemiological surveillance of AIVs was carried out in Eastern China,and one H11N2 subtype avian influenza virus,A/duck/Jiangxi/k0701/2009,was isolated from domestic duck in a live bird market.The analysis of complete genome sequences and genetic evolution showed that all eight segments of A/duck/Jiangxi/k0701/2009(H11N2) belong to Eurasian lineage,but genetic recombination was observed between A/duck/Jiangxi/k0701/2009(H11N2) and AIVs in some regions in Asia and Europe in varying degrees and extensively.Moreover,the PB2 and NS gene may be originated from A/mallard/Korea/GH170/2007(H7N7),NA gene may have the same origin with NA of HPAIV A/duck/Hebei/0710/2009(H5N2),and NP,PA,PB1 genes may have the same origin with A/Muscovy duck/Thailand/CU-LM1984/2009(H4N9).Our results indicated that A/duck/Jiangxi/k0701/2009 may be a multi-origin reassortant virus,and may play an important role in the evolution of HPAIVs.

Background: Although extensive data demonstrates that the majority of H6 duck isolates belonged to a single H6N2 virus lineage with a single gene constellation in southern China from 2000 to 2005, the prevalence of H6N2 virus in poultry in Eastern China is largely unknown.Results: Epidemiology revealed that H6N2 viruses were the most frequently detected influenza subtypes in live bird markets from 2002 to 2008 in Eastern China, but from 2009 onwards, they were replaced with novel H6N6 viruses. We phylogenetically and antigenically analyzed 42 H6 viruses isolated mainly in domestic ducks from 2002 to 2010 in Eastern China. Surprisingly, none of these isolates grouped with the previously described H6N2 viruses which belonged to a single H6N2 virus lineage with a single gene constellation in domestic ducks in southern China from 2000 to 2005. Two distinct hemagglutinin lineages were identified and they all underwent frequent reassortment with multiple virus subtypes from the natural gene pool, but few reassortants were persistent or prevalent.Conclusions: Five subtypes of H6 influenza viruses (H6N1, H6N2, H6N5, H6N6 and H6N8) cocirculated in Eastern China, which form a significant part of the natural influenza virus reservoir in domestic ducks, and significant viral reassortment is still ongoing in this species.

H5亚型高致病性禽流感在野生水禽中进化很慢,传给陆禽后其进化速度大大加快,当一些病毒在陆禽适应且毒力增强后又会反传给野生水禽。虽然野生水禽在感染高致病性禽流感后不能长久维持和储存病毒,但可以促进高致病性禽流感病毒远距离传播。与此同时,家养水禽在陆禽与野生水禽之间的流感病毒传播上起着非常重要的媒介和放大器作用,它们既可以携带多种亚型的低致病性禽流感病毒,又能长期储存陆禽来源的高致病性禽流感病毒,尤其是家鸭,无论是否免疫,抗体水平高低,都能带毒排毒,将高致病性禽流感病毒在野生水禽与陆禽间的传播放大。